Abstract HIV-1 packages two copies of genomic RNA (gRNA) as a dimer into newly formed viral particles. Retroviral assembly doesn?t depend on the presence of gRNA, yet gRNA packaging is essential for production of infectious virus particles. Robust and specific mechanisms must therefore be in place to ensure the genetic material is passed on to progeny virions. While there are numerous therapeutics in clinical use, there are currently no gRNA packaging or viral assembly inhibitors. Selective gRNA packaging is facilitated by interactions between the HIV- 1 Gag protein and conserved gRNA elements within the 5? untranslated region (5?UTR). Here, we focus on recent unexpected findings in HIV-1 RNA biology that revealed sequence heterogeneity at the 5? end of the HIV-1 RNA transcript. The variable number of G residues (1G, 2G and 3G) has been reported to affect the gRNA localization, with 1G RNA preferentially selected over 3G to be the viral genome. This is very surprising given that these two 9-kb RNAs only differ by 2 nucleotides. Preliminary data presented here strongly support the enrichment of 1G- containing transcripts among packaged gRNA. Importantly, various point mutants that abrogate the preference for 1G RNA have been identified. Exciting preliminary data support our major hypothesis that the ensemble of RNA structures adopted by the 5?UTR is significantly altered in 1G vs. 3G transcripts. These data provide a strong starting point for the proposed studies aimed at elucidating the mechanism by which transcriptional start site choice modulates gRNA packaging selectivity. More broadly, we will gain insights into how subtle sequence changes can alter the ensemble of 5?UTR RNA structures and impact viral replication fitness. The specific aims are: (1) To probe wild-type and mutant retroviral gRNA structure and dynamics; (2) To probe wild-type and mutant HIV-1 Gag RNA binding and packaging specificity. The results of these studies will help guide the design of novel therapeutic agents that target the 5?UTR and interfere with the essential conformational plasticity and/or key binding interactions with the expectation for a lower rate of mutational escape than conventional drugs.